Principles of single molecule localization microscopy. (a) Conventional fluorescence microscopy excites all fluorophores at once, and therefore the images of closely spaced fluorophores overlap. In this case, the best possible image resolution is around 250 nm when using visible light. (b) Single molecule localization microscopy (SMLM) techniques activate and observe only a sparse subset of fluorophores at any given time. Because the images of each fluorophore no longer overlap, the location of each fluorophore can be determined precisely. The fluorophore positions can be used to create a super-resolution image of the sample. Instead of plotting the diffraction-limited image of the fluorophore (top sequence), the measured location of each fluorophore is plotted (bottom sequence). (c) SMLM image of tagged chemotaxis receptors in Escherichia coli. Each small point is a single fluorophore with approximately 15-nm localization precision. The SMLM image is much sharper than the conventional image (inset in (c)). (d) The location of fluorophores can be determined more precisely if the fluorophore emits more photons. If the fluorophore only emits 100 photons (left) it becomes more difficult to locate the center of its image in comparison to emission of 1,000 (middle) or 10,000 (right) photons.